Note: Descriptions are shown in the official language in which they were submitted.
WO 95/20319 ~ ' 218 0 6 4 ~ pCT~S95/00989
SYNERGISTIC ANTIMICROBIAL COMPOSITIONS CONTAINING A
HALOGENATED ACETOPHENONE AND AN ORGANIC ACID
The present invention relates to certain compositions
t and processes useful for controlling the growth of one or
more microorganisms and for preventing spoilage caused by
bacteria and fungi in various products, materials or media,
particularly industrial products, materials or media. These
products, materials or media include wood pulp, wood chips,
lumber, adhesives, coatings, animal hides, paper mill
liquors, pharmaceutical formulations, cosmetics, toiletry
formulations, geological drilling lubricants, petrochemicals,
agrochemical compositions, paints, leather, wood,
metalworking fluids, cooling tower water, tanning liquors,
starch, proteinaceous materials, acrylic latex paint
emulsions, and textiles.
The novel compositions and processes incorporating the
compositions of the present invention show unexpected,
synergistic activity against microorganisms, including
bacteria and fungi. Specifically, the invention is directed
to the use of compositions and processes containing a halo-
genated acetophenone and at least one organic acid, its salt
or ester.
Many of the products, materials or media referred to
above, when wet or subjected to treatment in water are
susceptible to bacterial and/or fungal deterioration or
degradation unless steps are taken to inhibit such
degradation or deterioration. To control deterioration or
degradation caused by microorganisms, various industrial
microbicides are used, but some of these biocides are of
questionable utility because they have undesirable odors, are
high in cost, show a low degree of effectiveness and/or
create hazards with respect to storage, use and/or handling.
For instance, the use of such popular industrial
microbicides as organomercury compounds, organotin compounds
and chlorinated phenols has come under increasing regulatory
2180649
- 2/1 -
pressure in recent times because of their highly
toxicity and concern about their adverse effects on
the environment. Consequently, the industry has
continued to seek improved biocides that have low
toxicity and are capable of exhibiting prolonged
biocidal effect at normal use levels.
Japanese Patent Application 58-183,606 relates to
fungicide and algae-controlling agents containing a
mixture of an acetophenone compound with 3,5-
dimethy101,2,5-2H-tetrahydrothiadizine-2-thione;
methylenebisthiscyanate; 1,4-bis-(bromo-acetoxy)2-
butene; 1,2-bis-(bromoacetoxy)ethane; 2-bromo-2-nitro-
1,3-propanediol; or a mixture of a complex of 5-
chloro-2-methyl-4-isothiazdin-3-one and a metal salt
and a complex of 2-methyl-4-isothiazolin-3-ore and a
metal salt.
Organic acids can be used alone to control
microorganisms, and while some of these compounds are
generally regarded as safe, many of them have low
efficacy against bacteria and fungi unless extremely
high concentrations are used. In excessive
concentrations, these organic acids can be expensive
and could even be corrosive to certain industrial
materials. A method that can prevent excessive use of
organic acids and hence decrease cost is therefore
desirable.
Alternatively, halogenated acetophenones can be
used alone in low concentrations as low toxicity
biocides. However, at low concentrations, halogenated
acetophenones tend to have a narrow antimicrobial
spectrum and fail to completely prevent the growth of
microorganisms.
Accordingly, the present invention is directed to
microbicidal compositions and processes incorporating
these compositions that substantially obviate one or
more of the problems, limitations, and disadvantages
AMENDED SHEET
IPEA/EP
.~ . 2180649
- 2/2 -
of the prior art. In particular, the compositions of
the present invention are capable of controlling the
growth of at least one microorganism, especially fungi
or bacteria, over prolonged periods of time, and are
safe and economical to use. The present invention
also is directed to methods or processes of
controlling the growth of at least one microorganism.
The present invention provides a composition to
control the growth of at least one microorganism
comprising synergistically effective amounts of a
halogenated acetophenone and at least one organic
acid, its salt or ester. The composition provides
superior microbicidal
AN9ENDED SHEET
IPEA/EP
WO 95/20319 PCT/US95/00989
- ~~gp~49
activity at low concentrations against a wide range of
microorganisms.
The present invention also provides a method for
controlling the growth of at least one microorganism in or on
a material or medium susceptible to attack by the
microorganism which comprises the step of adding to the
material or medium a composition of the present invention
where the components of the composition are present in
synergistically effective amounts.
Furthermore, the present invention provides a method of
preventing spoilage of a product, material or medium caused
by bacteria and/or fungi comprising the step of adding to the
product, material or medium, a composition of the present
invention where the components of the composition are present
in synergistically effective amounts.
The synergistically effective amounts vary in accordance
with the material or medium to be treated and can, for a
particular application, be routinely determined without undue
experimentation by one skilled in the art.
The present invention also embodies the separate
addition of a halogenated acetophenone and at least one
organic acid, its salt or ester to the products, materials or
media described above. According to this embodiment, the
components are individually added to the system so that the
final amount of the halogenated acetophenone and at least one
organic acid, its salt or ester present at the time of use is
that synergistically effective amount required to control the
growth of at least one microorganism.
The compositions of the present invention are also
useful in preserving various types of industrial products,
media or materials susceptible to attack by microorganisms.
Such products, media or materials include but are not limited
to dyes, pastes, lumber, leather, textiles, pulp, wood chips,
tanning liquors, paper mill liquors, polymer emulsions,
2180649
WO 95/20319 PCT/I1S95/00989
- 4 -
paints, paper and other coating and sizing agents,
metalworking fluids, geological drilling lubricants,
petrochemicals, cooling water systems, pharmaceutical for-
mulations, cosmetics and toiletry formulations.
The compositions can also be useful in agrochemical
formulations for the purpose of protecting seeds or crops
against microbial spoilage.
Additional advantages of the invention will be set forth
in part in the description which follows, and in part will be
obvious from the description, or may be learned by the
practice of the invention. The advantages of the invention
may be realized and obtained by means of the elements and
combinations particularly pointed out in the claims.
It is to be understood that both the foregoing general
description and the following detailed description are
exemplary and explanatory only and are not restrictive of the
present invention as claimed.
When two chemical microbicides are combined into one
product or added separately three results are possible:
1) The chemicals in the product would produce an
additive (neutral) effect.
2) The chemicals in the product would produce an
antagonistic (negative) effect, or
3) The chemicals in the product would produce a
synergistic (positive) effect.
An additive effect has no economic advantage over the
individual components. The antagonistic effect would produce
a negative impact. Only a synergistic effect, which is less
likely than either an additive or antagonistic effect, would
produce a positive effect and therefore possess economic
advantages.
It is well-known in the microbicidal literature that
there is no theoretical method to provide the likelihood of
knowing, before actually testing, whether additive,
WO 95/20319 ~ 218 0 6 4 9 pCT~S95/00989
- 5 -
antagonistic or synergistic effects will be obtained when two
biocides are mixed to yield a formulation.
The microbicidal compositions combining halogenated
acetophenones and at least one organic acid, its salt or
ester demonstrate an unexpected, synergistic effect compared
to the respective components alone. Thus, these compositions
achieve superior, i.e., greater than additive, microbicidal
activity at low concentrations against a wide variety of
microorganisms. Examples of microorganisms include fungi and
bacteria such as, but not limited to, Trichoderma harzianum
and Pseudomonas aeruginosa. These two organisms are some of
the most common organisms associated with spoilage of
products, materials or media. Since these two are also some
of the toughest organisms to control, the composition of the
present invention is believed to be effective against most
bacteria and fungi. Preferably, the compositions of the
present invention have a low toxicity.
In accordance with the compositions of the present
invention, the halogenated acetophenone is preferably a
halogenated hydroxyacetophenone and more preferably has the
formula:
CoCN~X
Y
in which X is a halogen and Y is a halogen or H. Preferably
X is Br, F, C1 or I and Y is H, Br, F, C1 or I. Most
preferably, the halogenated acetophenone according to the
present invention is
2-bromo-4'-hydroxyacetophenone. The preparation of
monosubstituted 2-bromoacetophenones is described in U.S.
Patent No. 3,193,448, which disclosure is fully incorporated
by reference. 2-bromo-4'-hydroxyacetophenone or BHAP is
commercially available and is also easily synthesized from
commercially available raw materials.
WO 95/20319 , 218 0 6 4 9 pCT/US95/00989
- 6 -
The organic acids of the present invention are any
organic acids that produce a synergistic effect when combined
with the halogenated acetophenone. Examples of organic acids
include aromatic organic acids, cyclic organic acids,
aliphatic organic acids, their salts or esters. Specific
examples of effective organic acids, according to the
invention, include dehydroacetic acid, octanoic acid,
nonanoic acid, formic acid, sorbic acid, acetic acid, oxalic
acid, glycolic acid, citric acid, malic acid, propionic acid,
lauric acid, undecylenic acid, benzoic acid or derivatives of
benzoic acid such as 2-hydroxybenzoic acid, 3-hydroxybenzoic
acid or 4-hydroxybenzoic acid, methyl paraben or propyl
paraben.
The salts of the organic acids, preferably those
containing calcium, zinc, potassium, or sodium, may be used,
such as sodium benzoate or potassium sorbate. Preferred
esters are parabens such as methyl paraben and propyl
paraben.
In accordance with the present invention, mixtures of
these organic acids, salts or esters can also be used. When
such mixtures are used in combination with the halogenated
acetophenone, at least one of the organic acids in the
mixtures has a synergistic relationship with the halogenated
acetophenone. Organic acids, salts and esters useful in the
invention are commercially available or may be synthesized
from commercially available raw materials.
The organic acid may be chosen, for example, based on
the compatibility of the acid with the products, materials or
media. The compatibility is readily determined by adding the
organic acid to the products, material or media to be used.
Compatibility may be determined by criteria such as
solubility in a fluid system and/or lack of reactivity with
the fluid in question. When used in a fluid system, for
example, it is preferable that the organic acid be freely
2180649
WO 95/20319 PCT/US95/00989
_ 7 _
soluble or dispersible in the particular fluid system,
resulting in a uniform solution or dispersion. Examples of
fluid systems are tanning liquor, paper mill liquor, cooling
tower water, and paints.
In accordance with the present invention, the
composition may be in the form of a solid, dispersion,
emulsion or solution depending on the particular application.
Further, the components of the composition may be applied
separately or may be combined first and then applied to the
product, material or medium.
The composition of the present invention may be prepared
in liquid form by dissolving components (A) and (B) in an
organic solvent.
In the following discussion of preferred embodiments,
component (A) is 2-bromo-4'-hydroxyacetophenone (BHAP) and
component (B) is at least one organic acid, its salt or
ester.
As described above, components (A) and (B) of the
composition are used in synergistically effective amounts.
The weight ratios of (A) to (B) vary depending on the type of
microorganisms, as well as the products, material or media to
which the composition is applied. One skilled in the art can
readily determine the appropriate weight ratios for a
specific application.
In the present invention, the weight ratio of component
(A) to component (B) preferably ranges from about 0.01:99 to
about 99:0.01, more preferably from about 1:30 to about 30:1,
and most preferably from about 1:5 to about 5:1.
The following approximate range of weight ratios of BHAP
to the following organic acids are also preferred according
to the present invention:
BHAP:benzoic acid 1:1.7 to 1:333
BHAP:sodium benzoate 1:167 to 0.1:133
BHAP:sorbic acid 1:1.7 to 1:133
WO 95/20319 2 ~ 8 0 6 4 9 pCT/US95/00989
- 8 -
BHAP:potassium sorbate 1:33 to 1:267
BHAP:p-hydroxybenzoic acid 1:1.7 to 0.1:133
BHAP:dehydroacetic acid 1:6.7 to 1:67
BHAP:propionic acid 1:8 to 1:333
BHAP:methyl paraben 1:3 to 0.1:333
BHAP:propyl paraben 1:1.7 to 0.1:333
BHAP:nonanoic acid 1:17 to 1:133
BHAP:octanoic acid 1:1.7 to 1:133
BHAP:undecylenic acid 1:1.7 to 0.1:167
BHAP:lauric acid 1:1.7 to 1:333
BHAP:formic acid 1:1.7 to 1:333
BHAP:acetic acid 1:8 to 1:266
BHAP:oxalic acid 1:1.7 to 1:133
BHAP:citric acid 1:1.7 to 1:133
BHAP:malic acid 1:1.7 to 1:67
BHAP:glycolic acid 1:3 to 1:133
In general, an effective fungicidal and bactericidal
response can be obtained when the synergistic combination is
employed in concentrations ranging from about 0.01 to about
3000 ppm of 2-bromo-4-hydroxyacetophenone, preferably from
about 0.1 to about 1000 ppm, and most preferably from about
0.1 to about 500 ppm, and from about 0.1 ppm to about 1 % by
weight of the organic acid, preferably from about 0.1 to
about 5000 ppm, and most preferably from about 0.1 to about
2000 ppm.
The present invention also provides a method for
controlling the growth of at least one microorganism in or on
a material or medium susceptible to attack by the
microorganism which comprises the step of applying to the
material or medium a composition of the present invention
where the components of the composition are present in
synergistically effective amounts.
Furthermore, the present invention provides a method of
preventing spoilage of a product, material or medium caused
by bacteria and for fungi comprising the step of applying to
WO 95/20319 218 0 6 4 9 PCT/US95/00989
_ g _
said product, material or medium, a composition of the
present invention where the components of the composition are
present in synergistically effective amounts. For example,
the composition may be used to prevent the spoilage of seeds
or crops, such as cotton, barley, rice, maize, tobacco, etc.
The mode and rate of application of the composition
varies depending upon the intended use of the composition.
For instance, the composition may be applied by spraying or
brushing onto a material or product. The material or product
could also be treated by dipping in a suitable formulation of
the composition. In a liquid or liquid-like medium, the
composition could be added into the medium by pouring, or by
metering with a suitable device, so that a solution or a
dispersion containing the composition is produced. If used
as a liquid preservative, for example, the composition may be
prepared as an aqueous emulsion. If necessary, a surfactant
may be added to the composition.
In accordance with the invention, additional components
such as insecticides and the like may be added to the
foregoing preparations without affecting the synergistic
effects of the composition. Insecticides that may be used
include but are not limited to pyrethrins, nicotine,
chlordane, parathions, and methoxychlor.
The synergistic activity of the combinations described
above has been confirmed using standard laboratory
techniques, as illustrated below. The following examples are
intended to illustrate, not limit, the scope of the present
invention.
Microbiological evaluation
A. Fungal evaluation
A mineral salts-glucose medium was first prepared by
adding to 1 liter of deionized water: 0.7 g of KH2P04, 0.7
g of MgS04.7H20, 1.0 g of NH4N03, 0.005 g of NaCl, 0.002 g of
FeS04.7H20, 0.002 g of ZnS04.7H20, 0.001 g of MnS04.7H20, and
g of glucose. The pH of the medium was adjusted to 6 with
WO 95/20319 . 218 0 6 4 9 p~.~sg5/00989
- 10 -
1N NaOH. The medium was then dispensed into 5 mL amounts in
test tubes and autoclaved at 121°C for 20 minutes.
The fungus, Trichoderma harzianum, was grown on a potato
dextrose. agar slant for 7 to 10 days and a spore suspension
prepared by washing down the spores from the slant into a
sterile
saline solution. After the addition of biocides in the
desired concentrations to the sterile mineral salts-glucose
medium, the fungal spore suspension was added. The final
spore concentration was approximately 106cfu/mL. The
inoculated media was incubated at 28° C for 14 days.
B. Bacterial evaluation
Nutrient broth (2.5 g/liter of deionized water) was
prepared and the pH adjusted to 6 with 1N HCl. This was
dispensed in 5 mL amounts into test tubes and autoclaved for
20 minutes at 121 °C. After the addition of the biocides in
the desired concentrations to the nutrient broth, 100
microliters of a suspension of Pseudomonas aeruginosa cells
of approximately 9.3 x 108 cfc/mL were added and incubated at
37°C for 48 hours.
In Examples 1 through 19, a synergistic effect was
demonstrated by testing the combination of 2-bromo-4'-
hydroxyacetophenone (BHAP), designated as component A, and
the corresponding organic acid, its salt or ester, designated
as component B, in a series of tests in varying ratios and a
range of concentrations against the fungus Trichoderma
harzianum and also against the bacterium Pseudomonas
aeruginosa, using the methods described above.
For each component A and B in a mixture containing A and
B and for each component A and B acting alone, the lowest
concentration which completely prevented growth of the fungi
for two weeks and the bacteria for 48 hours was determined.
These concentrations were used as end points for synergism
calculations. End points for the components alone or in
mixtures described above were then compared with the end
PCTIUS95100989
W O 95/20319
- 11 -
points for the pure active ingredients alone in similarly
prepared flasks or test tubes.
Synergism was demonstrated by the method described by
Kull, E.C., Eisman, P.C., Sylwestrwicz, H.D., and Mayer, R.L.
1961. Applied Microbiology. 9: 538-541 wherein:
QA/Qa + QB/Qb is less than 1
Qa = Concentration of compound A in parts per million, acting
alone, which produced an end point.
Qb = Concentration of compound B in parts per million, acting
alone, which produced an end point.
QA = Concentration of compound A in parts per million, in the
mixture, which produced an end point.
QB = Concentration of compound B in parts per million, in the
mixture, which produced an end point.
When the sum of QA/Qa and QB/Qb is greater than one,
antagonism is indicated and when the sum is equal to one,
additivity is indicated. When the sum of this value is less
than one, synergism exists.
This procedure for demonstrating synergism of the
compositions of this invention is widely used and accepted.
More detailed information is provided in the article by Kull
et al. Further information concerning this procedure is
contained in U.S. Patent No. 3,231,509, which disclosure is
fully incorporated by reference.
The Examples demonstrate that in almost all of the
samples, the combination of BHAP with a corresponding organic
acid produced a synergistic result (indicated by a ratio
value of less than one.) There were a few samples, such as
in Table 2, <1.2 or <1.4, where synergistic results were
inconclusive because endpoints for the acids used alone were
not determined.
It will be apparent to those skilled in the art that
various modifications and variations can be made in the
compositions and methods of the present invention without
280649
WO 95/20319 - ~' ' PCT/LTS95/00989
- 12 -
departing from the spirit or scope of the invention. Thus,
it is intended that the present invention cover the
modifications and variations of this invention provided they
come within the scope of the appended claims and their
equivalents.
Z i 8 0 6 4 9 pCT/L1S95/00989
- WO 95/20319
- 13 -
Ezample 1
Component A = BHAP
Component B = Benzoic acid
Test organism Quantities producing end points (ppm)
Qa Qw Qv Qe Qn/Q. Qe/Qv Qw/Q.+
QB/Q6
Triclwderma 30 - - - _ _ _
harzianiun
- 7.5 - 250 0.25 0.5 0.75
- 15 - 25 0.5 0.05 0.55
- 15 - 50 0.5 0.1 0.6
- 15 - 100 0.5 0.2 0.7
- 15 - 250 0.5 0.5 1
_ _ 500 _ _ _ _
Pseudomonas 7.5 - - _ - - _
Qeraginosa
- 0.3 - 100 0.04 0.4 0.44
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 50 0.2 0.2 0.4
- 1.5 - 100 0.2 0.4 0.6
- 3 - 10 0.4 0.04 0.4.4
- 3 - 25 0.4 0.1 0.5
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
. - - - 250 - - _ _
WO 95/20319 2 i 8 0 6 4 9 pCT~S95100989
- 14 -
Example 2
Component A = BHAP
Component B = Sodium benzoate
Test organism Quantities producing end points (ppm)
b QB ~~~ QB~Qb
QB~Qb
Pseudomorras7.5 - - - - - -
aeruginosa
- 0.75 - 1000 0.1 1 < 1.1
- 1.5 - 1000 0.2 1 < 1.2
- 3 - 500 0.4 0.5 0.9
- 3 - 1000 0.4 1 < 1.4
- - > 1000 - - _ _
Note: Combinations against Trichoderma harzianum were not synergistic.
WO 95120319 , ' ~ ~ ~ PCTIUS95100989
- 15 -
Example 3
Component A = BHAP
Component B = Sorbic acid
Test organism Quantities producing end points (ppm)
Q. Qw Q. Qe QwIQ. QeIQ. QaIQ~+
QeIQ.
Trichoderma 30 - - - - _ _
hanianum
- 3 - 500 0.1 0.5 0.6
- 7.5 - 100 0.25 0.1 0.35
- 7.5 - 250 0.25 0.25 0.5
- 7.5 - 500 0.25 0.5 0.75
- 15 - 25 0.5 0.03 0.53
- 15 - SO 0.5 0.05 0.55
- IS - 100 0.5 0.1 0.6
- 15 - 250 0.5 0.25 0.75
- 15 - 500 0.5 0.5 I
_ _ 1000 _ - - _
Pseudomonar 7.5 - - - - - _
aeruginosa
- 0.3 - 100 0.04 0.4 0.44
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 50 0.2 0.2 0.4
- 1.5 - 100 0.2 0.4 0.6
- 3 - 10 0.4 0.04 0.44
- 3 - 25 0.4 0.1 0.5
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - - -
WO 95/20319 ~ . , ~ ~ ~ ~ p 6 4 9 pCT~S95100989
- 16 -
Example 4
Component A = BHAP
Component B = Potassium sorbate
Test organism Quantities producing end points (ppm)
Q. Qw Qb Qe ~/Q. QelQb
QB/Qb
T'richodermn30 - _ _ _ _ _
iiarzianum
- 7.5 - 2000 0.25 1 1.25
- 15 - 500 0.5 0.25 0.75
- 15 - 1000 0.5 0.5 < 1
15 2000 0.5 1 < 1.5
- - > 2000 - _ _ _
IItP' nmhrn~tinnc~n~incr
:~~..
.. ..b....r,. . .~cwsvawttw ucm~Ntv.lts wGIG 11VI J~'ItGI~IJUG.
WO 95/20319 ~ 218 0 6 4 9 pCT~S95/00989
..'.,
- 17 -
Example 5
Component A = BHAP
Component B = p-hydroxybenzoic acid
Test organism Quantities producing end points (ppm)
Q. ~ Qb Qe Q~/Q. Qs/Qb Q
QB/Qb
Trichodernsa 30 - - - - - -
ha~zianum
- 1.5 - 2000 0.05 1 < 1.05
- 3 - 2000 0.1 1 < 1.1
- 7.5 - 500 0.25 0.25 0.5
- 7.5 - 1000 0.25 0.5 0.75
- 15 - ZS 0.5 0.01 0.51
- 15 - 50 0.5 0.03 0.53
- 15 - 100 0.5 0.05 0.55
- 15 - 250 0.5 0.13 0.63
- IS - 500 0.5 0.25 0.75
_ - > 2000 - _ - --
Pseudomonas 7.5 - - - - - -
aeruginosa
- 0.3 - 250 0.04 0.5 0.54
- 0.75 - 100 0.1 0.2 0.3
- 0.75 - 250 0.1 0.5 0.6
- 1.5 - 50 0.2 0.1 0.3
- 1.5 - 100 0.2 4.2 0.4
- 1.5 - 250 0.2 0.5 0.7
- 3 - 25 0.4 0.05 0.45
- 3 - 50 0.4 0.1 0.5
- 3 - 100 0.4 0.2 0.6
- 3 - 250 0.4 0.5 0.9
_ _ 500 - _ _ _
WO 95/20319 ' 21 ~ 0 6 4 9 pCT~s95/00989
- 18 -
Example 6
Component A = BHAP
Component B = Dehydroacetic acid
Test organism Quantities producing end points (ppm)
Q1 QB ~/~ QB/Q6 KA/Qa+
QB/Q1
Trichoderma 30 - - - - - -
harzianurn i
- 7.5 - 100 0.25 0.2 0.45
- 7.5 - 250 0.25 0.5 0.75
- 15 - 100 0.5 0.2 0.7
- 15 - 250 0.5 0.5 1
_ _ 500 _ _ _ -
Pseudomonas 7.5 - - - - - -
aeruginosa
- 1.5 - 100 0.2 0.4 0.6
- 3 - 25 0.4 0.1 0.5
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - - -
WO 95/20319 PCT/US95/00989
=19 - 2 ~ so649
Example 7
Component A = BHAP -
Componeat B = Propionic acid
Test organism Quantities producing end points (ppm)
Q. ~ Qr Qe ~~Q. Qe~Qv Q
Qe~Qe
Trichoderma 30 - - - - - -
harzianum
- 7.5 - 1000 0.25 1 1.25
- 15 - 250 0.5 0.13 0.63
- 15 - 500 0.5 0.25 0.75
- 15 - 1000 0.5 0.5 < 1
_ _ 2000 _ _ _ -
Pseudomonas 7.5 - - - - -
aeruginosa
- 0.3 - 100 0.04 0.4 0.44
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 100 0.2 0.4 0.6
- 3 - 25 0.4 0.1 0.5
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - - -
WO 95!20319 218 0 6 4 9 pCT~S95100989
- 20
fixample 8
Component A = BHAP
Component B = Methyl paraben
Test organism Quantities producing end points (ppm)
Q. ~ Qv Qa Q~~~ Qe~Qe Qw~~'~
QB~Q6
Trichoderma 30 - - - -
harzianum
_ 3 - 500 0.1 0.5 0.6
_ 7,5 - 100 0.25 0.1 0.35
- 7.5 - 250 0.25 0.25 0.5
- 7.5 - 500 0.25 0.5 0.75
- 15 - 100 0.5 0.1 0.6
- 15 - 250 0.5 0.25 0.75
- 15 - 500 0.5 0.5 1
_ _ 1000 _ - _ __
Pseudomonas 7.5 - - - - -
aeruginosa
- 0.3 - 1000 0.04 1 < 1.04
- 0.75 - 1000 0.1 1 < 1.1
- I.5 - 500 0.2 0.5 0.7
- 1.5 - 1000 0.2 1 < 1.2
_ 3 - 10 0.4 0.01 0.41
- 3 - 25 0.4 0.03 0.43
_ 3 - 50 0.4 0.05 0.45
_ 3 - 100 0.4 0.1 0.5
- 3 - 250 0.4 0.25 0.65
- 3 - 500 0.4 0.5 0.9
- 3 - 1000 0.4 1 1.4
_ - > 1000 - - - _
1 4'.
.2
WO 95/20319 PCTIUS95l00989
- 21 - 2180649
Example 9
Component A = BHAP
Component B = Propyl paraben
Test organism Quantities producing end points (ppm)
QA Qb QB ~~~ QB~Qb ~~~+
QB~Qb
Trichoderma 30 - - - - - --
J~arzianum
- 1.5 - 250 0.05 0.5 0.55
- 3 - 250 0.1 0.5 0.6
- 7.5 - 100 0.25 0.2 0.45
- 7.5 - 250 0.25 0.5 0.75
- 15 - 50 0.5 0.1 0.6
- 15 - 100 0.5 0.2 0.7
- 15 - 250 0.5 0.5 1
_ - 500 _ _ - -
Pseudomonas 7.5 - - - - - -
aeraginosu
- 0.3 - 500 0.04 0.5 0.54
- 0.3 - 1000 0.04 1 < 1.04
- 0.75 - 500 0.1 0.5 0.6
- 1.5 - 250 0.2 0.25 0.45
- 1.5 - 500 0.2 0.5 0.7
- 3 - 5 0.4 0.01 0.41
- 3 - 10 0.4 0.01 0.41
- 3 - 25 0.4 0.03 0.43
- 3 - 50 0.4 0.05 0.45
- 3 - 100 0.4 0.1 0.5
i
- 3 - 250 0.4 0.25 0.65
- 3 - 500 0.4 0.5 0.9
- - >1~ - _ __ _
2180649
WO 95/20319 . PCT/US95/00989
i - 22 -
Ezample 10
Component A = BHAP
Component B = Nonanoic acid
Test organism Quantities producing end points (ppm)
Q, Qw Qb Q8 ~~~ QB~Qb Qw~'~+
QB/
Q6
Trichoderma 30 - - - - - -
harzianum
- 15 - 250 0.5 0.5 < 1
_ - 500 _ _ _ -
Pseudomonas 7.5 - - - - ' -
aeruginosa
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 100 0.2 0.4 0.6
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - - -
WO 95/20319 ~ ~ ~ ~ PCT/US95/00989
- 23 -
Example 11
Component I~ = BHAP
Component B = Octanoic acid
Test organism Quantities producing end points (ppm)
Q. Qw Qv Qe ~/~ Qe/Q~ Qw/~+
QB~Q~
Trichodernsa 30 - _ _ -.
ha~zianum
- 7.5 - 100 0.25 0.2 0.45
- 7.5 - 250 0.25 0.5 0.75
- 15 - 25 0.5 0.05 0.55
- 15 - 50 0.5 0.1 0.6
- 15 - 100 0.5 0.2 0.7
- 15 - 250 0.5 0.5 1
_ - 500 _ _ _ _
Pseudomonas 7.5 - - - - -
aeruginosa
- 1.5 - 100 0.2 0.4 0.6
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - _ _
WO 95/20319 ~ . 218 0 6 4 9 p~.~S95100989
- 24 -
Example 12
Component A = BHAI'
Component B = Undecylenic acid
Test organism Quantities producing end points (ppm)
Q. Qn Q~ Qe Qn~Q~ Qs~Q~ Qn~Q,+
Trichoderma 30 - - - - _ _
harzianum
- 1.5 - 1000 0.05 0.5 0.55
- 3 - 500 0.1 0.25 0.35
- 3 - 1000 0.1 0.5 0.6
- 7.5 - 250 0.25 0.13 0.38
- 7.5 - 500 0.25 0.25 0.5
- 7.5 - 1000 0.25 0.5 0.75
- 15 - 25 0.5 0.01 0.51
- 15 - 50 0.5 0.03 0.53
- 15 - 100 0.5 0.05 0.55
- 15 - 250 0.5 0.13 0.63
- 15 - 500 0.5 0.25 0.75
- 15 - 1000 0.5 0.5 1
- _ 2000 - _ _ _
Pseudomonar 7.5 - - - - _ _
aeruginosa
- 0.3 - 500 0.04 0.5 0.54
- 0.75 - 250 0.1 0.25 0.35
- 0.75 - 500 0.1 0.5 0.6
- 1.5 - 250 0.2 0.25 0.45
- 1.5 - 500 0.2 0.5 0.7
- 3 - 250 0.4 0.25 0.65
- 3 - 500 0.4 0.5 0.9
- - 1000 _ _ _ _
WO 95/20319 - ' ~ 218 D 6 4 9 pCT~S95/00989
- 25 -
Ezample 13
Component A = BHAP
Component B = Lauric acid
Test organism Quantities producing end points (ppm)
b QB ~/~ QB/Q6 QA/Qa+
I
QB/Qb
Trichodernia 30 - - - - _ _
jlaZlall1011
- 7.5 - 50 0.25 0.03 0.28
- 7.5 - 100 0.25 0.05 0.30
- 7.5 - 250 0.25 0.13 0.38
- 7.5 - 500 0.25 0.25 0.5
- 7.5 - 1000 0.25 0.5 0.75
- 7.5 - 2000 0.5 1 < 1.5
- 15 - 25 0.5 0.01 0.51
- ~15 - 50 0.5 0.03 0.53
- 15 - 100 0.5 0.05 0.55
- 15 - 250 0.5 0.13 0.63
- 15 - 500 0.5 0.25 0.75
- 15 - 1000 0.5 0.5 < 1
15 - 2000 0.5 1 < 1.5
_ - > 2000- - _ _
Pseudomonos 7.5 - - - _ _ _
aeruginosa
- 3 - 100 0.4 0.1 0.5
- 3 - 250 0.4 0.25 0.65
- 3 - 500 0.4 0.5 0.9
- 3 - 1000 0.4 1 < 1.4
_ _ > 1000- _ _ _
218 0 6 4 9 PCT/US95/00989
WO 95/20319
- 26 -
Example 14
Component A = BHAP
Component B = Formic acid
Test organism Quantities producing end points (ppm)
QI QB QA~~ QB~Qb
QB~Qb
Trichoderma 30 - - - - - -
I
- 3 - 1000 0.1 0.5 0.6
- 3 - 2000 0.1 1 < 1.1
- 7.5 - 250 0.25 0.13 0.38
- 7.5 - 500 0.25 0.25 0.5
- 7.5 - / 000 0.25 0.5 0.75
- 7.5 - 2000 0.25 1 < 1.25
- 15 - 25 0.5 0.01 0.51
- 15 - 50 0.5 0.03 0.53
- 15 - 100 0.5 0.05 0.55
- 15 - 250 0.5 0.13 0.63
- 15 - 500 0.5 0.25 0.75
- 15 - 1000 0.5 0.5 < 1
- _ > 2000- _ _ _
Pseudomonas 7.5 - - - - - -
aeruginosa
- 0.3 - 50 0.04 0.5 0.54
- 0.75 - 50 0.1 0.5 0.6
- 1.5 - 25 0.2 0.25 0.45
- 1.5 - 50 0.2 0.5 0.7
- 3 - 10 0.4 0.1 0.5
I
- 3 - 25 0.4 0.25 0.65
- 3 - 50 0.4 0.5 0.9
_ _ 100 _ _ _ _
-~ WO 95/20319 $ O ~ 4 7pCT/US95/00989
- 27 -
Example 15
Component A = BHAP
Component B = Acetic acid
Test organism Quantities producing end points (ppm)
Q1 QB QA/~ QB/QI QA/Qa
~'
Q8/Qb
Trichoderma 30 - - - -
- _
- 7.5 -- 2000 0.25 1 < 1.25
- 15 - 1000 0.5 0.5 < I
- 15 - 2000 0.5 1 < 1.5
- - > 2000- _ - _
Pseudomonas 7.5 - _ _ - - -
ocruginosa
- 0.75 - 50 0.1 0.5 0.6
- 1.5 - 50 0.2 0.5 0.7
- 3 - 25 0.4 0.25 0.65
- 3 - 50 0.4 0.5 0.9
- - 100 - _ _ _
. . 2180649
WO 95/20319 ~ . PCT/US95100989
- 28 -
Example 16
Component A = BHAP
Component B = Oxalic acid
Test organism Quantities producing end points (ppm)
Q. Q~ Q~ Qs ~/Q. Qe/Qv Q~/Q,+
QB/Q6
Trichoderma 30 - _ _ _
harzianum - _
- 15 - 1000 0.5 0.5 < 1
- 15 - 2000 0.5 1 < 1.5
- - > 2000- _ _ _
Pseudomonas 7.5 - _ - _ - _
aeruginosa
- 0.3 - 100 0.04 0.4 0.44
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 50 0.2 0.2 0.4
- 1.5 - 100 0.2 0.4 0.6
- 3 - 5 0.4 0.02 0.42
- 3 - 10 0.4 0.04 0.4.4
- 3 - 25 0.4 0.1 0.5
- - 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - _ _ _
i
z ~ so649
WO 95/20319 PCT/US95/00989
- 29 -
Example 17
Component A = BHAP
Component B = Citric acid
Test organism Quantities producing end points (o~cn)
Q. Qw Qb Qa Q~~Q. Qe~Qv Q,.~Q,+
. I
QB~Qb
Trichoderma 30 _ _ _ -
harzianurn - _
- 15 - 1000 0.5 0.5 < 1
- 15 - 2000 0.5 I < I.5
- > 2000- - _ _
Pseudomonas 7.5 - - - -
aeruginosa
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 100 0.2 0.4 0.6
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - _ _ _
i
2180649
WO 95/20319 PCT/US95/00989
- 30 -
Example 18
Comps cent A = BHAP
Component B = Malic acid
Test organism Quantities producing end points (ppm)
Qr Qe Qw/Q. Qe/Qr ~w/~'~
QB/Q6
Pseudontonar7.5 - - - _ _ _
aeruginosa
- 1.5 - 50 0.2 0.2 0.4
- 1.5 - 100 0.2 0.4 0.6
- 3 - 5 0.4 0.02 0.42
- 3 - 10 0.4 0.04 0.44
- 3 - 25 0.4 0.1 0.5
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - - -
Note: Combinations agauut rich erma~ianum were not synergistic.
_m 2180649
WO 95/20319 PCT/US95/00989
- 31 -
Example 19
Component A = BHAI?
Component B = Glycolic acid
Test organism Quantities producing end points (ppm)
Q. Qw Q~ Qe Qw~~ Qe~Q~ Qw~Q.'~
Qe~Qv
Pseudornonas 7.5 - _ _ _ - _
aeruginosa
- 0.75 - 50 0.1 0.2 0.3
- 0.75 - 100 0.1 0.4 0.5
- 1.5 - 50 0.2 0.2 0.4
- 1.5 - 100 0.2 0.4 0.6
- 3 - 10 0.4 0.04 0.44
- 3 - 25 0.4 0.1 0.5
- 3 - 50 0.4 0.2 0.6
- 3 - 100 0.4 0.4 0.8
- - 250 - - - -
Note: (:ombinations against Tridaodernra hmzimium were not synergistic.
